In the classic paradigm of central tolerance, thymocytes with specificity for self-antigens are deleted during development, thereby leaving the peripheral T cell repertoire devoid of any cells capable of reacting to self-tissues. While the significance of this mechanism in preventing autoimmunity is well established, growing evidence indicates that many self-antigen specific T cells routinely escape deletion and populate the peripheral T cell repertoire without causing disease. This is especially likely in the case of tissue-restricted self-antigens that are poorly expressed in the thymus. How peripheral self-antigen specific T cells remain tolerant in the presence of their cognate antigen poses an intriguing question as these cells are likely involved in autoimmunity as well as anti-tumor immunity. Due to the severe limitations of monoclonal TCR transgenic T cell systems to accurately model complex polyclonal populations of antigen-specific T cells, several fundamental questions about self-antigen specific T cells remain unresolved. First, it is unclear to what extent central tolerance mechanisms such as clonal deletion and regulatory T cell (Treg) development play in the regulation of T cell reactivity to tissue-restricted self-antigens. Furthermore, it is unclear whether self-antigen specific conventional T cells (Tconv) present in the periphery are simply ignorant of their antigen, functionally anergic, or actively suppressed by Treg cells. We have developed powerful experimental systems involving peptide:MHC tetramer-based cell enrichment techniques and tissue-restricted antigen transgenic mice that allow us to directly characterize rare polyclonal populations of self-antigen specific T cells that naturally arise in endogenous repertoires. These tools will enable us to investigate these issues at an unprecedented level of physiological significance. We hypothesize that deletional tolerance of self-antigen specific T cells is far less extensive than previously appreciated, particularly in th case of tissue-restricted self-antigens, and accordingly, the peripheral repertoire of T cells is normally populated with numerous potentially self-reactive clones. We believe that self-antigen specific CD4+ T cells preferentially adopt a Treg lineage fate during development, and these cells suppress their Tconv counterparts in the periphery to establish tolerance within the overall self-antigen specific population. We will test this hypothesis by pursuing the following initial specific aims: 1) Determine how thymic selection shapes the peripheral repertoire of CD4+ T cells specific for tissue- restricted self-antigens, and 2) Determine how steady state tolerance is maintained in peripheral populations of self-antigen specific CD4+ T cells. The achievement of these aims will greatly improve our understanding of how non-deletional mechanisms of peripheral T cell tolerance are normally established for self-antigens in the steady state, and how we may exploit this information for therapeutic purposes.